This chapter provides an overview of fluorescence lifetime measurements and their applications in biological imaging. It begins with an introduction to the photophysical processes involved, such as internal conversion, vibrational relaxation, fluorescence, intersystem crossing, and phosphorescence, and explains how these processes are characterized by decay rate constants. The focus is on fluorescence lifetime, which is an intrinsic property of fluorophores and is not affected by measurement methods or initial conditions. Fluorescence lifetime is sensitive to both internal factors (structure of the fluorophore) and external factors (temperature, polarity, and quenchers), making it a valuable tool for molecular imaging. The history of fluorescence lifetime imaging is traced back to the mid-19th century, highlighting early experiments with phosphorescence and the development of instruments like the phosphoroscope. The chapter also discusses the role of organic dye synthesis in expanding the range of fluorescent compounds and the theoretical foundations of fluorescence lifetime, including the Strickler-Berg equation. The techniques for measuring fluorescence lifetime, such as time-domain and frequency-domain methods, are described, along with the principles behind time-resolved fluorescence anisotropy and multiphoton excitation. The chapter further explores the theoretical aspects of fluorescence lifetime, including the influence of temperature, viscosity, and polarity on lifetime, and the mechanisms of internal quenching, such as internal rotation and excited state electron and proton transfer. Finally, the chapter discusses the practical applications of fluorescence lifetime imaging in various fields, including materials science, biology, and medicine, emphasizing its versatility and potential for advanced imaging techniques.This chapter provides an overview of fluorescence lifetime measurements and their applications in biological imaging. It begins with an introduction to the photophysical processes involved, such as internal conversion, vibrational relaxation, fluorescence, intersystem crossing, and phosphorescence, and explains how these processes are characterized by decay rate constants. The focus is on fluorescence lifetime, which is an intrinsic property of fluorophores and is not affected by measurement methods or initial conditions. Fluorescence lifetime is sensitive to both internal factors (structure of the fluorophore) and external factors (temperature, polarity, and quenchers), making it a valuable tool for molecular imaging. The history of fluorescence lifetime imaging is traced back to the mid-19th century, highlighting early experiments with phosphorescence and the development of instruments like the phosphoroscope. The chapter also discusses the role of organic dye synthesis in expanding the range of fluorescent compounds and the theoretical foundations of fluorescence lifetime, including the Strickler-Berg equation. The techniques for measuring fluorescence lifetime, such as time-domain and frequency-domain methods, are described, along with the principles behind time-resolved fluorescence anisotropy and multiphoton excitation. The chapter further explores the theoretical aspects of fluorescence lifetime, including the influence of temperature, viscosity, and polarity on lifetime, and the mechanisms of internal quenching, such as internal rotation and excited state electron and proton transfer. Finally, the chapter discusses the practical applications of fluorescence lifetime imaging in various fields, including materials science, biology, and medicine, emphasizing its versatility and potential for advanced imaging techniques.